Method of blocking immune complex binding to immunoglobulin Fc receptors

ABSTRACT

A method of modulating immune responses in mammals by blocking immune complex binding to immunoglobulin Fc receptors is described, comprising administering a peptide comprising a portion selected from the amino acid sequence 
     -Pro-Asp-Ala-Arg-His-Ser-Thr-Thr-Gln-Pro-Arg- 
     Specific uses in reducing immune complex mediated inflammation or tissue destruction and in reducing the human allergic response are disclosed.

This is a division of application Ser. No. 522,739, filed Aug. 12, 1983,now U.S. Pat. No. 4,579,840.

BACKGROUND OF THE INVENTION

Antibody synthesis is a defense response of higher vertebrates. Themolecular entities which stimulate antibody synthesis (e.g., a virusparticle) are called antigens. The introduction of an antigen into thebody of a higher vertebrate stimulates specific white blood cells, Blymphocytes, to produce antibodies that combine specifically with theantigen to prevent its further multiplication, or to otherwiseinactivate it. The study of antibodies and their action with antigens isa branch of immunology.

Antibodies which circulate in blood or other body fluids are termedhumoral antibodies, as distinguished from "membrane antibodies" whichremain bound to their parent lymphocytes. The term immunoglobulin isused to generically refer to all antibodies. In humans, allimmunoglobulins are divided into five classes termed IgG, IgA, IgM, IgDand IgE. Each immunoglobulin molecule consists of two pairs of identicalpolypeptide chains. The larger pair termed "heavy chains" and designatedgamma (γ), alpha (α), mu (μ), delta (δ) and epsilon (ε), respectively,are unique for each immunoglobulin class and are linked together bydisulfide (s--s) bonds between each chain. Each heavy chain consists ofabout 400 to 500 amino acid residues linked together by polypeptidebonds. Each light chain, by contrast, consists of about 200 amino acidsand are usually linked to a heavy chain by a single disulfide bond.

In 1969, Gerald Edelman first determined the amino acid sequence of anentire human IgG molecule. He found that both heavy and light chains areorganized into homology units or "domains" about 100 amino acids inlength. Subsequent sequence analysis of the other four immunoglobulinclasses demonstrate that they are also organized into structurallysimilar domains having different amino acid sequences. The first oraminoterminal domain of both light and heavy chains have discreteregions within which considerable variation in amino acids occur. Thesedomains are therefore termed variable (V) domains and are designatedV_(H) in heavy chains and V_(L) in light chains.

The molecular association of a V_(L) and V_(H) domain within an intactimmunoglobulin forms an antigencombining site which may bind to aspecific antigen with high affinity. The domain structure of all lightchains is identical regardless of the associated heavy chain class. Eachlight chain has two domains, one V_(L) domain and one domain with arelatively invariant amino acid sequence termed constant, light orC_(L).

Heavy chains, by contrast may have either three (IgG, IgA, IgD) or four(IgM, IgE) constant or C domains termed C_(H) 1, C_(H) 2, C_(H) 3, andC_(H) 4 and one variable domain, termed V_(H). Alternatively, C domainsmay be designated according to their heavy chain class; thus C.sub.ε 4indicates the C_(H) 4 domain of the IgE (epsilon) heavy chain.

Visualization of antibodies by electron microscopy or by x-raydiffraction reveals that they have a "Y" shape. IgA and IgM antibodies,in addition, combine in groups of two and five, respectively, to formdimers and pentamers of the basic Y shaped antibody monomer. See FIG. 1.

When antibodies are exposed to proteolytic enzymes such as papain orpepsin, several major fragments are produced. The fragments which retainantigen-binding ability consist of the two "arms" of the antibody's Yconfiguration and are termed Fab (fragment-antigen binding) or Fab'2which represent two Fab arms linked by disulfide bonds. The other majorfragment produced constitutes the single "tail" or central axis of the Yand is termed Fc (fragmentcrystalline) for its propensity to crystallizefrom solution. The Fc fragment of IgG, A, M, and D consists of dimers ofthe two carboxy-terminal domains of each antibody (i.e., C_(H) 2 andC_(H) 3 in IgG, IgA and IgD, and C_(H) 3 and C_(H) 4 in IgM.) The IgE Fcfragment, by contrast, consists of a dimer of its three-carboxyterminalheavy chain domains (C.sub.ε 2, C.sub.ε 3 and C.sub.ε 4).

The Fc fragment contains the antibody's biologically "active sites"which enable the antibody to "communicate" with other immune systemmolecules or cells and thereby activate and regulate immune systemdefensive functions. Such communication occurs when active sites withinantibody regions bind to molecules termed Fc receptors. See FIG. 2A.

Fc receptors are molecules which bind with high affinity and specificityto molecular active sites within immunoglobulin Fc regions. Fc receptorsmay exist as integral membrane proteins within a cell's outer plasmamembrane or may exist as free, "soluble" molecules which freelycirculate in blood plasma or other body fluids.

Each of the five antibody classes have several types of Fc receptorswhich specifically bind to Fc regions of a particular class and performdistinct functions. Thus IgE Fc receptors bind with high affinity toonly IgE Fc regions or to isolated IgE Fc fragments. It is known thatdifferent types of classspecific Fc receptors exist which recognize andbind to different locations within the Fc region. For example, certainIgG Fc receptors bind exclusively to the second constant domain of IgG(C_(H) 2), while Fc receptors mediating other immune functions bindexclusively to IgG's third constant domain (C_(H) 3). Other IgG Fcreceptors bind to active sites located in both C_(H) 2 and C_(H) 3domains and are unable to bind to a single, isolated domain.

Once activated by antibody Fc region active sites, Fc receptors mediatea variety of important immune killing and regulatory functions. CertainIgG Fc receptors, for example, mediate direct killing of cells to whichantibody has bound via its Fab arms (antibody-dependent cell mediatedcytotoxicity--(ADCC)). Other IgG Fc receptors, when occupied by IgG,stimulate certain white blood cells to engulf and destroy bacteria,viruses, cancer cells or other entities by a process known asphagocytosis. Fc receptors on certain types of white blood cells knownas B lymphocytes regulate their growth and development intoantibody-secreting plasma cells. Fc receptors for IgE located on certainwhite cells known as basophils and mast cells, when occupied byantigenbridged IgE, trigger allergic reactions characteristic ofhayfever and asthma.

Certain soluble Fc receptors which are part of the blood complementsystem trigger inflammatory responses able to kill bacteria, viruses andcancer cells. Other Fc receptors stimulate certain white blood cells tosecrete powerful regulatory or cytotoxic molecules known generically aslymphokines which aid in immune defense. These are only a fewrepresentative examples of the immune activities mediated by antibody Fcreceptors.

It is only after antibodies bind to antigen or are otherwise caused toaggregate that active sites within the Fc region are able to bind to andactivate Fc receptors. Fc receptors are, therefore, the critical linkbetween antibodies and the remainder of the immune system. Fc receptorbinding to antibody Fc region active sites may thus be characterized asthe "final common pathway" by which antibody functions are mediated. Ifan antigen-bound antibody does not bind to an Fc receptor, the antibodyis unable to activate the other portions of the immune system and istherefore rendered functionally inactive.

Any peptide with the ability to bind to immunoglobulin Fc receptors hastherapeutic usefulness as an immunoregulator by virtue of the peptide'sability to regulate binding to the receptor. Such an Fc receptor"blocker" occupies the immunoglobulinbinding site of the Fc receptor andthus "short circuits" the immunoglobulin's activating ability. FIG. 2B.

SUMMARY OF THE INVENTION

Most of the amino acids which make up antibodies function as molecular"scaffolding" which determine the antibody's structure, a highly regularthree-dimensional shape. It is this scaffolding which performs thecritical function of properly exposing and spatially positioningantibody active sites which consist of several amino acid clusters. Aparticular active site, depending upon its function, may already beexposed and, therefore, able to bind to cellular receptors.Alternatively, a particular active site may be hidden until the antibodybinds to an antigen, whereupon the scaffolding changes orientation andsubsequently exposes the antibody's active site. The exposed active sitethen binds to its specific Fc receptor located either on a cell'ssurface or as part of a soluble molecule (e.g., complement) andsubsequently triggers a specific immune activity.

Since the function of an antibody's "scaffolding" is to hold andposition its active sites for binding to cells or soluble molecules, theantibody's active sites, when isolated and synthesized as peptides, canperform the immunoregulatory functions of the entire antibody molecule.

Depending upon the particular type of Fc receptor to which an activesite peptide binds, the peptide may either stimulate or inhibit immunefunctions. Stimulation may occur if the Fc receptor is of the type thatbecomes activated by the act of binding to an Fc region or,alternatively, if an Fc active site peptide stimulates the receptor. Thetype of stimulation produced may include, but is not limited to,functions directly or indirectly mediated by antibody Fc region-Fcreceptor binding. Examples of such functions include, but are notlimited to, stimulation of phagocytosis by certain classes of whiteblood cells (polymorphonuclear neutrophils, monocytes and macrophages;macrophage activation, antibody-dependent cell mediated cytotoxicity(ADCC); natural killer (NK) cell activity; growth and development of Band T lymphocytes and secretion by lymphocytes of lymphokines (moleculeswith killing or immunoregulatory activities).

The ability to stimulate immune system functions, including those listedabove, is known to be therapeutically useful in treating diseases suchas infectious diseases caused by bacteria, viruses or fungi, conditionsin which the immune system is deficient due either to congenital oracquired conditions, cancer and many other afflictions of human beingsor animals. Such immunostimulation is also useful to boost the body'sprotective cellular and antibody response to certain injected or orallyadministered substances administered as vaccines. This list is notintended to be all inclusive and merely provides representative examplesof diseases or conditions in which immune stimulation has establishedtherapeutic usefulness.

Inhibition of immune system functions may occur if an active sitepeptide binds to a particular Fc receptor which is not activated by themere act of binding to an Fc region. Such Fc receptors normally become"activated" only when several Fc regions within an antigen-antibodyaggregate or immune complex simultaneously bind to several Fc receptors,causing them to become "crosslinked". Such Fc receptor crosslinking byseveral Fc regions appears to be the critical signal required toactivate certain types of Fc receptors. By binding to and blocking suchan Fc receptor, an active site peptide will prevent Fc regions withinimmune complexes or antigen-antibody aggregates from binding to thereceptor, thus blocking Fc receptor activation. See FIG. 2B.

The ability to inhibit immune system functions is known to betherapeutically useful in treating diseases such as allergies,autoimmune diseases including rheumatoid arthritis and systemic lupuserythematosis, certain types of kidney diseases, inflammatory boweldiseases such as ulcerative colitis and regional enteritis (Crohn'sdisease), certain types of inflammatory lung diseases such as idiopathicpulmonary fibrosis and hypersensitivity pneumonitis, certain types ofdemylinating neurologic diseases such as multiple sclerosis, autoimmunehemolytic anemias, idiopathic (autoimmune) thrombocytopenic purpura,certain types of endocrinological diseases such as Grave's disease orHashimoto's thyroiditis and certain types of cardiac disease such asrheumatic fever. Immunosuppression is also therapeutically useful inpreventing the harmful immune "rejection" response which occurs withorgan transplantation or in transplantation of bone marrow cells used totreat certain leukemias or aplastic anemias. This list is not intendedto be all inclusive but merely provides representative examples ofdiseases or conditions in which immunosuppression is known to betherapeutically useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of antigens bound to an antibody. Thelight and heavy chains of the antibody's Fab arms, and the biologicallyactive sites of the Fc region are shown.

FIG. 2(A) is a schematic illustration of an antibody bound, via its Fcregion active site, to a cellular Fc receptor FIG. 2(B) is a schematicillustration of active site peptides which bind to and block, theantibody's Fc receptor, thus preventing the antibody's immunologicalactivity. Such Fc receptor blockage forms the basis of the presentinvention.

FIG. 3 is a schematic illustrating an immune complex bound, via its Fcregion active sites, to two cellular Fc receptors

FIG. 4 is a schematic illustrating active site peptides which bind toand block cellular Fc receptors, thus preventing the immune complex'simmunological activity. Such Fc receptor blockage forms the basis of thepresent invention.

DETAILED DESCRIPTION OF KNOWN ACTIVE SITE PEPTIDES

In the following sections, the amino acid components of the peptides areidentified as abbreviations for convenience. These abbreviations areintended to include both the D- and L-forms although the L-form ispreferred:

    ______________________________________                                        Amino Acid        Abbreviation                                                ______________________________________                                        L-glycine         GLY                                                         L-alanine         ALA                                                         L-valine          VAL                                                         L-leucine         LEU                                                         L-isoleucine      ILE                                                         L-proline         PRO                                                         L-methionine      MET                                                         L-cysteine        CYS                                                         L-phenylalanine   PHE                                                         L-tyrosine        TYR                                                         L-tryptophan      TRP                                                         L-histadine       HIS                                                         L-lysine          LYS                                                         L-arginine        ARG                                                         L-aspartic acid   ASP                                                         L-asparagine      ASN                                                         L-glutamic acid   GLU                                                         L-glutamine       GLN                                                         L-serine          SER                                                         L-threonine       THR                                                         L-ornithine       ORN                                                         Carbobenzoxyl-amino acid                                                                        Z-amino acid                                                N--acetyl-amino acid                                                                            AC-amino acid                                               ______________________________________                                    

I. Complement Blocking Peptides

The classical pathway for complement activation consists of a group ofblood plasma proteins which are activated to form a biochemical cascadebeginning with the first complement component, C1 and ending with acellular "attack complex" consisting of C5, C6, C7, C8 and C9. Thepattern of activation is highly specific and begins when C1 binds to Fcregion active sites within antibody-antigen complexes of either IgG orIgM. A molecular subunit of C1, termed C1q, contains six identical Fcreceptors for IgG or IgM. C1q is an example of a "soluble" Fc receptorthat exerts its bioactivity (complement activation resulting in celllysis and inflammation) without being anchored to a cell surfacemembrane. The amino acid residues which contribute to the Fc active sitefor C1q in human IgG have been localized to the C_(H) 2 domain. Johnsonand Thames (J. Immunol., 117, 1491 (1975)) and Boackle, Johnson andCaughman (Nature, 282, 742 (1979)) found that peptides with sequencesderived from the C_(H) 2 of human IgGl at aa 274-281(Lys-Phe-Asn-Trp-Tyr-Val-AspGly) had substantial complement activatingability when the peptides were adsorbed to erythrocytes. In particular,one peptide with the aa (amino acid sequence(Lys-Ala-Asp-Trp-Tyr-Val-Asp-Gly) was about as effective in activatingC1q-mediated cell lysis as immune complexes formed by heat aggregatedIgG. The aforementioned researchers attributed this activity to thepeptide's ability to act as an active binding site for the C1q Fcreceptor. Other synthetic peptides with sequences derived from thisregion of IgG or from the aa 487-491 region of C_(H) 4 of IgM(Glu-Trp-Met-Gln-Arg), e.g. ##STR1## were less active, while Trp-Leu andTyr-Glu-Ala-Gly were inactive.

Subsequently, Prystowsky, et al. (Biochemistry, 20, 6349 (1981)), andLukas, et al. (J. Immunol., 127, 2555 (1981)) demonstrated that peptidesfrom an immediately adjacent C_(H) 2 region from aa 281 to 292 wereinhibitors of C1-mediated hemolysis. Specifically, peptides identical toIgG, C_(H) 2 residues 281-290 (Gly-Val-Gln-Val-His-Asn-Ala-Lys-Thr-Lys)and aa282-292 (Val-Gln-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-OH) wereapproximately as active as inhibitors as intact monomeric IgG. Otherpeptides, viz. aa275- 290(Phe-Asn-Trp-Tyr-Val-Asp-Gly-Val-Gln-Val-His-Asn-Ala-Lys-Thr-Lys),aa275-279 (Ac-Phe-Asn-Trp-Tyr-Val), aa289-292 (Thr-Lys-Pro-Arg) wereless active.

Other investgators (Burton, et al., Nature, 288, 338 (1980), using othercriteria, have concluded that the IgG, C1q-binding site is located amongC_(H) 2 residues aa316-340. They have, however, not reported oncomplement blocking activities of peptides from this

II. Immunostimulatory Active Site Peptides from IgG

A. Tuftsin

Tuftsin is a tetrapeptide, with sequence Thr-Lys-Pro-Arg, and is presentin the second constant domain of all human IgG subclasses and in guineapig IgG at aa 289-292. It was originally isolated from proteolyticdigests of IgG by Najjar who found it to stimulate phagocytosis bygranulocytes, monocytes and macrophages in vitro and is described inU.S. Pat. No. 3,778,426. Subsequent studies have shown Tuftsin to beactive at nanomolar concentrations in many species including humans,cows, dogs, rabbits, guinea pigs and mice. In addition to itsphagocytosis stimulating ability, Tuftsin has been shown to stimulateADCC, Natural Killer (NK) cell activity, macrophage-dependent-T-celleducation and antibody synthesis to T-cell-dependent and independentantigens in vitro and in vivo. Tuftsin is believed to act by binding tostereospecific receptors on granulocytes, macrophages and lymphocytes.Analysis of these receptors indicate that they resemble IgG Fc receptorsin both number and ligand dissociation constants. Recent studies byRatcliffe and Stanworth (Immunol. Lett., 4, 215 (1982)) and ourselvesdemonstrate that Tuftsin does bind to IgG Fc receptors since itcompetitively inhibits human IgG binding to human monocyte IgG Fcreceptors. Tuftsin's immunostimulatory abilities are thus attributed toits ability to bind to immunoglobulin Fc receptors.

B. Rigin

Rigin is a tetrapeptide analogue of Tuftsin located in the peptideregion of human IgG which spans the C_(H) 2 and C_(H) 3 domains at aa341-345 having sequence Gly-Gln-Pro-Arg. (Veretennikova, et al., Int. J.Peptide Protein Res., 17, 430 (1981)). It has phagocytosis-stimulatingabilities similar to those of Tuftsin and is described in U.S. Pat. No.4,353,823.

C. Twenty-Four Residue Immunostimulatory Peptide

In 1982, Morgan, Huguli and Weigle (Proc. Natl. Acad. Sci. USA, 79, 5388(1982)) reported the sequence of a 24 residue peptide identical to IgGaa 335-358 with the ability to nonspecifically activate lymphocytes. Thepeptide was shown to induce polyclonal B cell proliferation,antigen-specific antibody responses and Natural Killer (NK)cell-mediated lysis. This peptide(Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Ser-Arg-Glu-Glu-Met)and the 23 residue peptide lacking the carboxy-terminal methionineprobably acts by binding to lymphocyte Fc receptors for IgG.

III. IgG-Derived Fc Receptor Blocking Peptides

A. Decapeptide--From C_(H) 3 of Human IgG

In 1975, Ciccimarra, et al. (Proc. Natl. Acad. Sci. USA, 72, 2081(1975)) reported the sequence of a decapeptide from human IgG whichcould block IgG binding to human monocyte IgG Fc receptors. This peptideis identical to IgG aa 407-416(Tyr-Ser-Lys-Leu-Thr-Val-Asp-Lys-Ser-Arg). Stanworth, by contrast, wasnot able to demonstrate that this peptide could block monocyte IgGbinding. He did, however, show that the peptide blocked human IgGbinding to macrophage IgG Fc receptors of mice (Stanworth, Mol.Immunol., 19, 1245 (1982)).

B. Heptapeptide from C_(H) 2 of Human IgG

In 1982, Ratcliffe and Stanworth (Immunol. Lett., 4, 215 (1982))demonstrated that a peptide identical to IgG aa 295-301(Gln-Tyr-Asp-Ser-Thr-Tyr-Arg) could slightly block IgG binding to humanmonocyte IgG Fc receptors. By contrast, a related peptide identical toIgG, C_(H) 2, residues at aa 289-301 had no monocyte IgG blockingactivity.

IV. IgE Fc Receptor Blocking Peptides

A. Human IgE Pentapeptide (HEPP)

In 1975, Hamburger reported that a pentapeptide with sequence derivedfrom human IgE C.sub.ε 3 at aa 320-324 (Asp-Ser-Asp-Pro-Arg) couldinhibit a local cutaneous allergic reaction (Prausnitz-Kustner) byapproximately 90%. (Hamburger, Science, 189, 389 (1975) and U.S. Pat.Nos. 4,171,299 and 4,161,322.) This peptide has subsequently been shownto inhibit systemic allergic disease in humans after injection by thesubcutaneous route. Recent studies demonstrate that the peptide hassignificant affinity for the IgE Fc receptor of human basophils and canblock human IgE binding to basophil IgE Fc receptors by up to 70%(Plummer, et al., Fed. Proc., 42, 713 (1983)). The observed ability ofthis peptide to block systemic allergic disease in humans is attributedto the peptide's ability to bind to cellular IgE Fc receptors.(Hamburger, Adv. Allergology Immunol. (Pergamon Press: New York, 1980),pp. 591-593.

B. Hexapeptide from Human IgE

In 1979 Hamburger reported that a hexapeptide derived from C.sub.ε 4 ataa 476-481 (Pro-Asp-Ala-Arg-His-Ser) could block human IgE-binding toIgE Fc receptors on a human lymphoblastoid cell line (wil-2wt)(Hamburger, Immunology, 38, 781 (1979)). This peptide had beenpreviously implicated as an agent useful in blocking IgE-binding tohuman basophil IgE Fc receptors (U.S. Pat. No. 4,161,522).

V. Other Biologically Active Peptides from Human IgE

In 1982, Stanworth (Mol. Immunol., 19, 1245 (1982)) reported that adecapeptide with sequence identical to a portion of C.sub.ε 4 of humanIgE at aa 505515 (Val-Phe-Ser-Arg-Leu-Glu-Val-Thr-Arg-Ala-Glu) caused amarked enhancement of binding of ¹²⁵ I-human IgG to mouse macrophages.Interaction of this peptide with Fc receptors, however, was notdemonstrated.

In 1979 Stanworth, et al. demonstrated that certain peptides withsequences identical to portions of C.sub.ε 4 of human IgE, viz. aa495-506 (Pro-Arg-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe) and smallerderivatives thereof were able to cause degranulation of human and rodentmast cells and thus might be useful in allergic desensitization therapy.(Biochem, J., 180, 665 (1979); Biochem, J., 181, 623 (1979); andEuropean Patent Publication EP No. 0000252). No evidence was presented,however, that these peptides acted by virtue of binding toimmunoglobulin Fc receptors.

VI. Attempts to Discover IgG, Fc Receptor-Blockers for Neutrophils

Past attempts to isolate or synthesize peptides able to block IgG Fcreceptors on Neutrophils have uniformly failed. Studies in which IgG wasenzymatically degraded or otherwise chemically cleaved and resultantfragments tested for Fc receptor blocking activity demonstrated that nofragment smaller than an intact Fc fragment had measurable Fc receptorinteraction. (Barnett-Foster, et al., J. Immunol, 120, 407 (1978);Barnett-Foster, et al., Mol. Immunol., 19, 407 (1982)).

VII. Attempts to Discover IgE, Fc Receptor-Blockers for Monocyte orMacrophages

Past attempts to isolate or synthesize peptides able to block IgE Fcreceptors on monocytes/macrophages have uniformly failed. Studies inwhich IgE was enzymatically degraded or otherwise chemically cleaved andresultant fragments tested for Fc receptor blocking activitydemonstrated that no fragment smaller than an intact Fc fragment hadmeasurable IgE Fc receptor interaction. (Takatsu, et al., J. Immunol.,114, 1838 (1975); Dorrington, et al., Immunol. Rev., 41, 3 (1978);Perez-Montfort, et al., Mol. Immunol. 19, 1113 (1982)).

DETAILED DESCRIPTION OF THE INVENTION

This invention describes the sequences of new and useful peptides thatcan block the binding of human IgG immune complexes to IgG Fc receptorson human polymorphonuclear neutrophils (PMNs), of IgG and IgE immunecomplexes to IgG and IgE Fc receptors on monocytes and macrophages (MMs)and other white blood cells.

A further object is to describe how such new and useful peptides andtheir analogs and derivatives may be used to treat certain humandiseases in which immune complexes contribute to disease pathogenesis.Such diseases include, but are not limited to, rheumatoid arthritis,systemic lupus erythematosus, glomerulonephritis, serum sickness,polyarteritic nodosa and other forms of vasculitis, and other diseasesdescribed under the generic category of "autoimmune diseases",idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, asthma andother diseases and conditions.

A still further object is to describe how such peptides can preventdestruction of erythrocytes (red blood cells) and platelets (bloodclotting cells) as occurs in certain autoimmune diseases such asautoimmune hemolytic anemias and idiopathic thrombocytopenic purpura,respectively.

A still further object is to describe how certain of these peptides bindto Fc receptors on other classes of white blood cells (lymphocytes andbasophils) which enable such peptides to perform additionaltherapeutically useful functions.

Other objects and advantages of the invention will be apparent to thosein the art from the preceding and following description of theinvention.

PMNs and MMs As Mediators of Immune Complex-Induced Inflammation

It is generally recognized that PMNs and MMs (monocytes are immaturemacrophages) accumulate in great numbers at the sites of most acuteimmunologically induced tissue injury. It has been recently appreciatedthat these cells are not mere passive "bystanders" in the inflammatoryprocess, but rather directly contribute to tissue inflammation anddestruction. PMNs contain cellular organelles known as lysosomes whichcontain proteolytic enzymes and inflammatory mediators sequestered in alatent, inactive form. Under certain conditions, PMNs and MMs arestimulated to release the contents of their lysosomes to the externalcellular millieu, resulting in considerable tissue inflammation and celldeath. Such inflammation and cell death are mediated by a variety ofbiologically active substances. The lysosomal granules of PMNs and MMscontain many different proteolytic and glycolytic enzymes (viz.collagenase which degrades collagen, elastase which degrades elastin,lysozyme which degrades certain types of carbohydrate polymers, etc.)which cause direct destruction of cartilage, connective tissue andcells. Certain of these enzymes may also activate the blood clottingsystem or inflammatory peptide mediators such as bradykinin orcomplement. Other important inflammatory mediators released by PMNs andMMs include prostaglandins (PGs) and leukotrienes (LTs) which have avariety of potent inflammatory and immunomodulatory effects. Some ofthese effects, together with the elicting substance are enumeratedbelow.

(1) Prostaglandin E2 (PGE2)--PGE2 produces vasodialation, erythema,increased vascular permeability, edema (swelling), potentiates theinflammatory and pain producing actions of histamine and bradykinin andstimulates bacterial endotoxin-induced collagenase production byleukocytes. (Kuehl, et al., Science, 210, 978 (1980)).

PGE2 also has potent immunosuppressive effects and can suppress NK andADCC-mediated killing of cancer cells, T cell colony growth, clonalproliferation of the committed granulocyte-macrophage stem cell andantigen and mitogen-induced B-lymphocyte maturation intoantibody-secreting plasma cells. PGE2 also directly activatesshort-lived T suppressor lymphocytes which can, in turn, suppress otherprotective immune functions. Such PGE2-mediated suppression of theimmune system is thought to be important in producing theimmunosuppression that frequently accompanies various forms of cancer.(Goodwin, Clin. Immunol. Immunopath., 15, 106 (1980); Stenson, et al.,Immunol. 125, 1 (1980); Oroller, et al., Cell. Immunol., 39, 165 (1978);Leung, et al., J. Immunol., 129, 1742 (1982); Fischer, et al., J.Immunol., 126, 1452 (1981); Klein, et al., Immunol., 48, 337 (1983);Goodwin, et al., Cancer Immunol. Immunother., 8, 3 (1980)).

(2) Leukotrienes C4 (LTC4), D4 (LTD4) and E4 (LTE4)--These leukotrienes,in various combinations, constitute the slow reacting substance ofanaphylaxis (SRS-A) thought to be important in the pathogenesis of manyinflammatory and allergic diseases, especially asthma. Theseleukotrienes are hundreds to thousands of times more potent, on a molarbasis, than histamine in eliciting inflammation and pulmonarybronchoconstriction. (Samuelsson, Science, 220, 568 (1983)).

(3) Leukotriene B4 (LTB4)--This leukotriene is one of the most potentchemotatic substances known for certain human leukocytes. It is producedby preparations of human peripheral leukocytes, neutrophils, lung tissueand other cells. LTB4 attracts neutrophils, and eosinophils, both ofwhich are present in high numbers at sites of inflammation.

LTB4 also stimulates the release of lysosomal enzymes, includinglysozyme, from neutrophils which directly mediates tissue destruction.LTB4 is probably involved in the pathogenesis of many inflammatoryconditions, including rheumatoid arthritis in which LTB4 is found inelevated concentrations in affected joints. (Samuelsson, Science, 220,568 (1983); Science, 215, 1382 (1982)).

Immune complexes also cause PMNs and MMs to release highly reactive freeradicals such as the superoxide anion (O₂ --) which directly damagestissue which it contacts. (Weiss, et al., J. Immunol., 129, 309 (1982);Fantone, J. Pathol., 107, 397 (1982)).

Of particular importance to this invention is the fact that IgG and IgEcontaining immune complexes, aggregates or objects to which IgG or IgEis attached via Fab-mediated binding or by passive surface adsorption("complexes") are potent stimulators of phagocytosis (engulfment anddigestion of complexes, particulate matter and cells) by PMNs and MMsand subsequent enzyme and inflammatory mediator release. Suchstimulation is known to depend on the binding of IgG or IgE Fc regionswithin immune complexes to Fc receptors located on the PMN or MM cellsurface.

It is known that the exact molecular form in which Fc regions arepresented to PMN Fc receptors (e.g., IgG or IgE antigen complexes, heataggregated IgG or IgE, IgG or IgE passively adsorbed to cell orparticulate surfaces) is not important in triggering lysosomal enzyme orinflammatory mediator release. The critical factor in common to thesestimulatory forms of IgG or IgE is that multiple Fc regions in a fixed,relatively immobilized form be present to simultaneously bind tomultiple PMN or MM Fc receptors. Such multiply engaged Fc receptors thentrigger lysosomal enzyme or inflammatory mediator release.

Examples of Disease Processes in Which Immune Complex-Mediated LysosomalEnzyme or Inflammatory Mediator Release Contribute to Inflammation andTissue Destruction

I. Rheumatoid Arthritis (RA)

Lesions of RA are thought to first develop within joint spaces. An asyet unidentified agent or condition triggers a local, intraarticularsynthesis of IgG and IgM antibodies directed toward the Fc region ofIgG. Such "rheumatoid factors (RFs)", being a type of immune complex,accumulate within the joint and bind to Fc receptors of leukocytes,including PMNs and of the complement system. Such binding triggers aninitial inflammatory reaction which attracts blood-born leukocytes,especially PMNs. PMNs in large numbers migrate into the joint space and,there, encounter RF immune complexes which trigger lysosomal enzymesecretion and subsequent cartilage and tissue destruction. While otherleukocytes including monocytes and macrophages also contribute to theinflammation by a similar process, PMNs frequently constitute the greatmajority of cells present and often exceed 25,000 PMNs per cubicmillimeter. RFs also activate the complement system which, inconjunction with PMNs and other cells and molecules, produce the tissuedestruction characteristic of the disease. (Perez, et al. in Textbook ofRheumatology, Vol. 1 (W. B. Saunders: Philadelphia, 1981), pp. 179-194.)Some of the peptides detailed in the present invention can block IgGimmune complex binding to PMNs, monocytes and macrophages and canthereby reduce or prevent inflammation and tissue destruction ofrheumatoid arthritis and other immune complex-mediated inflammation.

II. Idiopathic Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a condition in which the normallythin, gas permeable wall of the lung's respiratory unit, the alveolus,is greatly thickened and replaced with large amounts of relativelygas-impermeable, fibrous connective tissue. This greatly reduces thelung's ability to respire and may lead to chronic pulmonaryincapacitation and death. IPF frequently accompanies idiopathicinterstitial pneumonias and the interstitial pneumonias of rheumatoidarthritis, systemic lupus erythematosus, scleroderma andpolymyositis-dermatomyositis. (Dreisin, et al., N. Engl. J. Med. 298,353 (1978)). The final common pathway leading to the tissue destructionin IPF is believed to involve IgG-immune complexes produced eithersystemically or locally within the lung parenchyma. (Lawrence, et al.,N. Engl. J. Med. 302, 1187 (1980)). Localization of immune complexeswithin the lung leads to an influx of PMNs and monocytes from bloodwhich accumulate in large numbers within the interstitium and withinalveolar structures. Monocytes then develop into mature macrophages andjoin the normally present pulmonary macrophages. IgG Fc regions withinthe immune complexes then combine with PMN and MM Fc receptors causinglysosomal enzyme and inflammatory mediator release, inflammation andalveolar destruction. (Hunninghake, et al., Clin. Immunol. Rev., 1(3),337 (1981-1982)). Over a period of time, this process results in"scarring" and generalized pulmonary fibrosis. Some of the peptidesdetailed in the present invention can block IgG-immune complex bindingto IgG Fc receptors on PMNs, monocytes and macrophages and can therebyreduce or prevent inflammation and tissue destruction of idiopathicpulmonary fibrosis and other immune complex-mediated diseases.

III. Immune Complex-Induced Glomerulonephritis

The glomeruli of kidneys are the filtration devices which separate fromblood the plasma ultrafiltrate that ultimately becomes urine. Glomeruliare easily damaged by the inflammatory processes initiated by immunecomplexes which accumulate as a result of the blood filtration process.At an early stage of immune complex-induced glomerular injury, monocytesand macrophages accumulate in the glomerular mesangium where theyencounter immune complexes. IgG Fc regions within these complexes bindto IgG Fc receptors of the monocytes and macrophages and are therebystimulated to release lysosomal enzymes and the inflammatory mediatorspreviously discussed. These substances produce glomerular inflammation,(glomerulonephritis) which may lead to kidney failure and subsequentdeath. (Holdsworth, J. Immunol., 130, 735 (1983); Striker, J. Exp. Med.149, 127 (1979); Hunsicker, J. Exp. Med., 150, 413 (1979)). Immunecomplex-mediated glomerulonephritis and resultant kidney failure, forexample, is the single leading cause of death in patients with systemiclupus erythematosis. (Kumar in Pathologic Basis of Disease, eds. S. L.Robbins and R. S. Cotran (W. B. Saunders: Philadelphia, 1979), p. 304.)Many other conditions such as rheumatoid arthritis, other autoimmunediseases, infectious diseases such as streptococoal or hepatitis virusinfection and others are accompanied by glomerulonephritis caused byimmune complexes. Some of the peptides detailed in the present inventioncan block IgG immune complex binding to monocyte and macrophage IgG Fcreceptors and can thereby reduce or prevent inflammation and tissuedestruction of immune complex-mediated glomerulonephritis.

IV. Immune Complex Mediated Lung-Inflammation

of Hypersensitivity Pneumonitis

Hypersensitivity Pneumonitis (HP) includes a spectrum of conditionscharacterized by granulomatous interstitial and alveolar-filling lungdiseases associated with exposure to a wide range of inhaled organicdusts and particles. Affected individuals synthesize relatively largeamounts of IgG directed against the offending inhaled dust and produceIgG immune complexes within the lung parenchyma. These complexes bind toIgG Fc receptors of PMNs, monocytes and pulmonary macrophages which, ina manner similar to that previously discussed, are stimulated to releaselysosomal enzymes and inflammatory mediators which produce an acutepneumonia. If this process is continued for a period of time, the lungdamage may become permanent in the form of chronic granulomatousinterstitial pneumonitis. (Stankus, Allergologie, 4, 8 (1981)). Some ofthe peptides detailed in the present invention can block IgG-immunecomplex binding to IgG Fc receptors of PMNs, monocytes and macrophagesand can thereby reduce or prevent inflammation and tissue destruction ofhypersensitivity pneumonitis and other immune-complex mediated diseases.

V. IgE-Immune Complex-Mediated Inflammation in Asthma

Atopic (IgE-mediated) asthma is an inflammatory lung disease in whichIgE bound to pulmonary mast cells and circulating basophil Fc receptorscauses them to release inflammatory mediators upon exposure to thesensitizing allergan (antigen). It is also known that IgE not alreadybound to cellular Fc receptors may also bind to the sensitizing allerganto form IgE-allergan immune complexes. These circulating IgE immunecomplexes may then bind to monocyte or macrophage IgE Fc receptorscausing them to release the various inflammatory mediators previouslydiscussed. Additionally, IgG directed against the sensitizing allerganmay be present and may also produce IgG-allergan immune complexes. Thesecomplexes may then bind to IgG Fc receptors on PMNs and monocytes andmacrophages in the lungs and thereby contribute to lysosomal enzyme andinflammatory mediator release. Some of the peptides detailed in thepresent invention can block IgE-immune complex binding to IgE Fcreceptors on monocytes and macrophages and can thereby reduce or preventinflammation characteristic of asthma and other disease pathogenesis.(Melewicz, et al., Clin. Exp. Immunol., 49, 364 (1982); Spiegelberg, etal., 42, 124 (1983); Scott, et al., Fed. Proc., 42, 129 (1983)).

Examples of Other Immune Diseases

I. Autoimmune Hemolytic Anemias

This autoimmune disease arises when certain immune system cellsrecognize antigens on erythrocytes (red blood cells) as foreign andcause the synthesis of antibodies (usually IgG) directed toward theerythrocytes. This "anti-erythrocyte Ig" then binds to the erythrocytevia its Fab antigen-binding arms, leaving the Fc region exposed to theerythrocyte exterior. Macrophages, and to a lesser extent monocytes thenbind to such IgG-sensitized erythrocytes via cell surface IgG Fcreceptors. Most such binding occurs in the spleen and results inphagocytosis and destruction of erythrocytes and subsequent anemia.

Some of the peptides detailed in the present invention can block IgGreceptors on monocytes and macrophages and can therefore reduce orprevent IgG Fc-mediated destruction of erythrocytes and subsequentanemia. (Engelfriet, et al. in A Seminar on Immunemediated celldestruction (American Association of Blood Banks: Washington, D.C.), pp.93-130 (1981).

II. Idiopathic (Autoimmune) Thrombocytopenic Purpura (ITP).

ITP is a syndrome characterized by chronic thrombocytopenia (lowplatelet count) caused by a circulating antiplatelet antibody thatresults in platelet destruction by phagocytic leukocytes. In mostpatients, the antibody is of the IgG class and is directed toward anormally present platelet-associated antigen. When IgG-coated plateletsencounter macrophages and monocytes, especially in the spleen, theexposed IgG Fc regions on the platelets bind to IgG Fc receptors onmacrophages and monocytes and stimulate platelet phagocytosis anddestruction. (McMillan, N. Engl. J. Med., 304, 1135 (1981)). Agentswhich block macrophage and monocyte IgG Fc receptors are known to beefficacious in treating this condition. For example, Fehr, et al. (N.Engl. J. Med., 306, 1254 (1982)) and Imbach, et al. (Lancet, June 6,1981, p. 1228) demonstrated that intervenous, monomeric IgG administeredto patients with ITP significantly reduced platelet destruction. Theyattributed the observed efficacy to blockade of IgG Fc receptors ofmacrophages and other phagocytic leukocytes. Some of the peptidesdetailed in the present invention can block macrophage and monocyte IgGFc receptors and thus represent a significant improvement overintervenous IgG since peptides are relatively inexpensive to produce anddo not carry with them the risk of infectious disease transmission(e.g., hepatitis) which accompanies human blood products.

Peptides Which Block Immune Complex Binding to Fc Receptors May Also ActAs Immunostimulants

In addition reducing or preventing immune-complex-mediated inflammationand tissue destruction, the peptides disclosed in this invention mayhave other important effects on the immune system which may prove to betherapeutically efficacious in treating human diseases.

As described earlier in this disclosure, when IgG complexes bind tomonocyte IgG Fc receptors, monocytes secrete prostaglandin E2 (PGE2)which inhibits many important immune defense functions. These inhibitedfunctions include Natural Killer (NK) cell and ADCC killing of cancercells, growth and development of T lymphocytes, andgranulocyte-macrophage stem cells and antigen and mitogen-inducedB-lymphocyte maturation into protective antibody-secreting plasma cells.Additionally, PGE2 also directly activates short-lived suppressorlymphocytes which may in turn, suppress other protective immunefunctions. The peptides described in this invention which block IgGimmune complexes to monocyte IgG Fc receptors may, therefore be expectedto act as immunostimulants by virtue of their ability to inhibit immunecomplex-mediated PGE 2 secretion.

Some of the peptides described in the present invention also demonstratethe ability to block IgG and/or IgE immune complex binding to lymphocyteFc receptors. Such abilities may be expected to be therapeuticallyuseful in several ways.

I. Immunostimulation

All antibodies are synthesized by plasma cells which develop from Blymphocytes. B lymphocyte-to-plasma cell development is, therefore,critical for the production of protective antibody synthesis. Substanceswhich inhibit this development may result in an inadequate concentrationof antibodies and may render a person susceptible to infection. Ofparticular importance to the present invention is the fact thatcomplexes or aggregates of antigen and antibody, termed immunecomplexes, are potent inhibitors of B lymphocyte development. Suchinhibition is thought to occur by several mechanisms, all of whichrequire that Fc regions of antibodies within the immune complex bind tolymphocyte Fc receptors.

One well characterized mechanism of immune complex-induced B-lymphocytesuppression occurs when IgG Fc regions within an immune complex bind toIgG Fc receptors on B lymphocytes. Because such an immune complexcontains two or more IgG Fc regions, two or more Fc receptors may besimultaneously bound and thus "crosslinked" by virtue of theantigen-antibody bridge (FIG. 3). It is believed that this crosslinkingof Fc receptors directly triggers the B lymphocyte inhibition(Oberbarnscheidt, et al., Immunol., 35, 151 (1978); Kolsch, et al.,Immunol. Rev., 49, 61 (1980)). Some of the peptides detailed in thepresent invention can bind to B lymphocyte Fc receptors and thus preventimmune complexes from binding. Because peptides do not cause Fc receptorcrosslinking, they can prevent immune complex-mediated inhibition of Blymphocyte development (FIG. 4). The clinical effect of such a peptidewould be to cause stimulation of protective antibody synthesis.

Another mechanism whereby IgG or IgE Fc receptor-blocking peptides maystimulate antibody synthesis involves immunoglobulin-binding factors(IBFs). IBFs are soluble Fc receptors released from activated Tlymphocytes, PMNs and possibly monocytes and macrophages. IgG IBF is apotent suppressor of B lymphocyte development into antibody-secretingplasma cells.

In order to produce such suppression, it is believed that IgG IBF mustfirst bind to Fc regions of IgG molecules and to molecular structures onthe surface of B lymphocytes. It is known that IgG IBF-mediatedsuppression is abrogated if IBF is prevented from binding to IgG Fcregions. (Fridman, et al., Immunol. Rev., 56, 51 (1981); Bich-Thuy, J.Immunol., 129, 150 (1982)). It is therefore expected that peptides whichbind to and thus block IBF Fc receptors should also block IBF-mediated Blymphocyte suppression. These peptides should cause stimulation ofprotective antibody synthesis.

A third mechanism whereby peptides of the present invention maystimulate immune responsiveness involves peptide modulation of asubstance known as T cell Replacing Factor (TRF). TRF is secreted bycertain T lymphocytes ("helper T cells") under activating conditionssuch as exposure to antigen or artificial mitogens. TRF then binds toreceptors on B lymphocytes which, in turn, triggers development intoplasma cells. There is considerable evidence that the TRF receptor alsobinds to IgG Fc regions and therefore is also an Fc receptor. TRF isbelieved to stimulate B lymphocyte development by binding to the TRF/Fcreceptor and thus preventing IgG immune complexes from binding to thesame receptor. By this mechanism, TRF directly stimulates B lymphocytedevelopment and prevents immune complexes from inhibiting development.(Kolsch, et al., Immunol. Rev., 49, 35 (1980)). Some of the peptidesdescribed in the present disclosure can, like TRF, bind to B lymphocyteFc receptors and are therefore expected to share some of TRFsimmunostimulatory activities.

II. Immunomodulation

Interactions between the various leukocytes which constitute thecellular arm of the immune system are necessary for normal immunefunction. The induction of most immune responses is triggered byinteractions between T lymphocytes and antigen-presenting cells. Duringthis process, different subsets of T lymphocytes proliferate anddifferentiate into functionally distinct cells with helper, suppressor,inducer or killer activities. These cells may then, in turn, directlyparticipate in immune defense or may activate the defensive functions ofother leukocytes such as B lymphocytes or monocytes/macrophages.

An in vitro counterpart of these in vivo events is the autologous mixedlymphocyte reaction (AMLR). In the AMLR, normal T lymphocytes arestimulated to proliferate when co-cultured with autologous non-Tlymphocytes including monocytes/macrophages and B lymphocytes. Thisreaction is thought to approximate many of the cellular processes thatoccur during a normal immune response. (Smolen, et al., J. Immunol.,129, 1050 (1982); Goeken, et al., Hum. Immunol., 6, 79 (1983)).

Of particular importance to the present invention is the fact that IgGimmune complexes are potent inhibitors of the AMLR. Immune complexes arethought to directly exert their inhibitory effects on T lymphocytes bybinding to IgG Fc receptors of the T lymphocytes which normallyparticipate in the AMLR reaction. (Kabelitz, et al., Eur. J. Immunol.,12, 687 (1982)).

The AMLR is greatly suppressed in patients with a variety of autoimmuneor neoplastic diseases including systemic lupus erythematosus (Sakane,et al., Proc. Natl. Acad. Sci. U.S.A., 75, 3464 (1978)), Sjogren'ssyndrome (Miyasaka, et al., J. Clin. Invest., 66, 928 (1980)), primarybiliary cirrhosis (James, et al., J. Clin. Invest., 66, 1305 (1980)),Hodgkin's lymphoma (Engleman, et al., J. Clin. Invest., 66, 149 (1980)),and chronic lymphocytic leukemia (Smith, et al., J. Natl. Cancer Inst.,58, 579 (1977)). This suppression is thought to be due, in part, to theinhibitory effects of IgG immune complexes on normal T lymphocytefunction. Some of the peptides disclosed in the present invention canblock IgG immune complex binding to T lymphocytes and can thereforeblock immune-complex-induced inhibition which is thought to be importantin the pathogenesis of the above conditions.

Peptides which block immune complex binding to Fc receptors may also beexpected to stimulate cellular or delayed type hypersensitivity (DTH)which is known to be important in defense against cancer and certaininfectious diseases such as tuberculosis. Immune complexes cansignificantly inhibit DTH in experiments using mice (Douvas, Ann.Immunol. Inst. Pasteur, 132C, 307 (1981)). Such inhibition is known tobe dependent on the presence of Fc regions within the immune complexwhich bind to cellular Fc receptors.

III. Immunoinhibition

Antibody-dependent cell-mediated cytotoxity (ADCC) is a process by whichT lymphocytes, monocytes/macrophages and polymorphonuclear neutrophilsdestroy foreign or infectious cells. IgG antibodies must first bind toantigens on the target cell which sensitizes the cell for recognition byADCC cells. Upon encounter with an IgG-sensitized target, IgG Fcreceptors on the ADCC cell bind to exposed Fc regions on the surface ofthe target cell. Such Fc receptor binding activates the ADCC cell todirectly lyse the target cell, causing its death.

Inappropriate cell killing via ADCC is thought to mediate some of theinflammation and organ destruction which occurs in chronic activehepatitis (Cochrane, et al., Lancet, 1, 441 (1976)), Ulcerative colitis(Hibi, et al., Clin. Exp. Immunol., 49, 75 (1982)). Hashimoto'sthyroiditis (Calder, et al., Clin. Exp. Immunol., 14, 153 (1973)), andother conditions. Some of the peptides disclosed in the presentinvention can block lymphocyte IgG Fc receptors and are therefore usefulto block ADCC-mediated killing. Such inhibition is expected to preventmuch of the inflammation and tissue destruction characteristic of theabove diseases.

Peptides Which Exert A Direct Anti-Allergic Effect

Certain of the peptides described in this invention have antiallergicactivities, in addition to the effects previously described. It is knownthat IgE-mediated allergies (viz. allergic rhinitis (hayfever), types ofasthma, allergic reactions to insect stings) occur by a mechanism inwhich IgE is bound, via its Fc region, to IgE Fc receptors located onmast cells and basophils. When the offending allergan (the antigen whichoriginally elicited IgE synthesis) is presented to such sensitized mastcells and basophils and binds to the cell-bound IgE, inflammatorymediators are released which produce the immediate allergic reactioncharacteristic of allergies. If the sensitizing IgE is prevented frombinding to mast cell or basophil IgE Fc receptors, however, mediatorrelease does not occur. It is known that mast cell and basophil IgE Fcreceptors may be blocked by administering either chemically isolated IgEFc fragments or certain peptides that are described in the United Statesand foreign patents discussed above. Such compounds have affinity forthe IgE Fc receptor and thus bind to it and prevent IgE from binding. Inthis manner, the allergic response can be abrogated independent of theparticular antigen which elicited IgE synthesis. (Hamburger, Science,189, 389 (1975); Hamburger, Adv. Allergology Immunol. (Pergamon Press:New York, 1980), pp. 591-593; Plummer, et al., Fed. Proc., 42, 713(1983)).

Certain of the peptides described in the present invention can bind toIgE Fc receptors on human basophils and can block subsequent IgEbinding. These peptides therefore have antiallergic properties which maybe useful in the treatment of allergy in animals and in humans.

ACTIVE SITE PEPTIDES OF THE INVENTION

As indicated above, this invention is concerned with a method ofblocking immune-complex-mediated inflammation, new peptides havingtherapeutic value in various areas, therapeutic compositions containingthese peptides, and methods for use thereof.

The present invention provides active site peptides having the followingsequences:

I. A peptide having the amino acid sequence

A-Pro-Asp-Ala-Arg-His-Ser-Thr-Thr-Gln-Pro-Arg-B,

wherein A is a substituent selected from the group consisting of

H-,

Gln-,

Leu-Gln-,

Val-Leu-Gln-, and

Glu-Val-Leu-Gln-;

and wherein B is a substituent selected from the group consisting of

-OH,

-Lys,

-Lys-Thr, and

-Lys-Thr-Lys,

II. A peptide having the amino acid sequence

C-Thr-Thr-Gln-Pro-Arg-D,

wherein C is a substituent selected from the group consisting of

H-,

Ser-,

His-Ser-,

Arg-His-Ser-,

Ala-Arg-His-Ser-, and

Asp-Ala-Arg-His-Ser-;

and wherein D is a substituent selected from the group consisting of

-OH,

-Lys,

-Lys-Thr, and

-Lys-Thr-Lys.

III. A peptide having the amino acid sequence

Thr-Gln-Pro-Arg-E,

wherein E is a substituent selected from the group consisting of

-OH,

-Lys,

-Lys-Thr, and

-Lys-Thr-Lys,

IV. A peptide selected from the group consisting of

Arg-His-Ser-Thr-Thr-F

Ala-Arg-His-Ser-G,

Asp-Ala-Arg-His-J, and

K-Pro-Asp-Ala-Arg,

wherein F is a substituent selected from the group consisting of

-OH,

-Gln,

-Gln-Pro,

-Gln-Pro-Arg,

-Gln-Pro-Arg-Lys,

-Gln-Pro-Arg-Lys-Thr, and

-Gln-Pro-Arg-Lys-Thr-Lys;

wherein G is a substituent selected from the group consisting of

-OH

-Thr, and

-Thr-Thr-F;

wherein J is a substituent selected from the group consisting of

-OH,

-Ser, and

-Ser-G;

and wherein K is a substituent selected from the group consisting of

H-,

Leu-,

Gln-Leu,

Val-Gln-Leu, and

Glu-Val-Gln-Leu.

It is to be considered that the scope of the present invention isinclusive of the unsubstituent peptides as well as those which areterminally substituted by one or more functional groups which do notsubstantially affect the biological activity disclosed herein. From thisstatement it will be understood that these functional groups includesuch normal substitution as acylation on the free amino group andamidation on the free carboxylic acid group, as well as the substitutionof amino acids such as the D-isomers of the naturally occuring aminoacids. The peptides of this invention are highly unusual since they areable to bind to and block cellular Fc receptors in the same manner asthe much larger parent IgG or IgE molecules, a portion of which theyresemble. It is especially unusual and surprising that some of thesepeptides can bind to and block (i) IgG receptors of PMNs; (ii) IgG andIgE receptors of lymphocytes; and (iii) IgE receptors of monocytes sincescientific investigators skilled in the art described herein haveconcluded in the recent scientific literature that appreciable Fcreceptor binding, as described in examples 1-3, requires, at minimum, anintact Fc region of the IgG and IgE molecules. (Barnett-Foster, et al.,Mol. Immunol., 19, 407 (1982); Barnett-Foster, et al., J. Immunol., 120,407 (1978); Perez-Montfort, et al., Mol. Immunol., 19, 1113 (1982);Dorrington, et al., Immunol. Re., 41, 3 (1978); Takatsu, et al., J.Immunol., 114, 1838 (1975); Seiler, et al., Immunobiol., 158, 254(1981)).

It is believed therefore that the activity requirements of the moleculesare dictated by each molecule's sterochemistry, that is, the particular"folding" of the molecule. In this regard, it should be understood thatpolypeptide bonds are not rigid but flexible, thus allowing polypeptidesto exist as sheets, helices, and the like. As a result, the moleculesare flexible and will "fold" in a certain way. In the present inventionit has been discovered that the peptides "fold" in the same manner asthe long chain parent polypeptide and therefore exhibit the samebiological characteristics. For this reason, the peptides may besubstituted by various functional groups so long as the substituents donot substantially affect the biological activity or interfere with thenatural "folds" of the molecule.

The ability of the peptides to retain their biological activity andnatural folding is illustrated by the fact that they have Fc receptorbinding activity similar to the parent antibody molecules. While thepeptides of the present invention are believed to act by "blocking" Fcreceptor activity as described herein, it is not intended that thepresent invention be limited to any particular mechanism of action.

The Fc receptor blocking activity of the subject peptides was assessedusing well established techniques and procedures. In particular, arosette assay was employed which uses either monomeric, chemically orheat aggregated human IgG or IgE obtained from human myeloma sera.(Gonzalez-Molina, et al., J. Clin. Invest., 59, 616 (1977); Spiegelberg,et al. in Immunoassays: Clinical Laboratory Techniques for the 1980's(Alan R. Liss: New York, 1980), pp. 287-300). The IgG or IgE is thenadsorbed to ox-erythrocytes which has been previously treated withtrypsin and pyruvic aldehyde. Fresh human cell PMNs, monocytes andbasophils from a patient with basophilic chronic myelogenous leukemia orhuman cell lines (RPMI-8866, and Daudi for lymphocyte Fc receptors andU937 for monocyte/macrophage Fc receptors) were then incubated in thepresence or absence of inhibitors (monomeric IgG or IgE or peptides) atequal concentrations for fifteen minutes before addition of theindicator oxerythrocytes. In the absence of inhibitors, the cellsexpressing surface Fc receptors formed rosettes, clusters of three ormore indicator erythrocytes that bound to the cells by virtue of exposedFc regions on the erythrocyte surface. Addition of inhibitor myelomaproteins or inhibitor peptides produced a reproducable reduction ofrosette formation, calculated by dividing the percentage of rosettesformed when the diluent, phosphate buffered isotonic saline, was usedalone as control and then multiplying the calculated quotient by 100.

Table 1 presents the inhibition observed by representative peptideinhibitors calculated as a percentage of the inhibition observed withintact, monomeric myeloma proteins. Thus, 50 percent inhibition implysthat the peptide was 50% as inhibitory as the intact myeloma protein towhich it was compared.

                                      TABLE 1                                     __________________________________________________________________________    ROSETTE INHIBITION EXPRESSED AS PERCENT INHIBITION BY MONOMERIC IgG or        IgE                                                                                                      MONOCYTE/                                                           NEUTROPHIL                                                                              MACROPHAGE                                                                             LYMPHOCYTE                                                                             BASOPHIL                                          (PMN) Fc Receptor                                                                       Fc Receptor                                                                            Fc Receptor                                                                            Fc Receptor                      PEPTIDE          IgG       IgG  IgE IgG  IgE IgG IgE                          __________________________________________________________________________    Thr--Val--Leu--His--Gln--Asn--                                                                 88         74  95  34    95  78 100                          Trp--Leu--Asp--Gly--Lys                                                       Ac--Gln--Pro--Glu--Asn                                                                         95         19  55  94   NS   74  89                          Pro--Asp--Ala--Arg--His--Ser                                                                   95        100  87  95    98 105 101                          Thr--Thr--Gln--Pro--Arg                                                                        NS        NS   58  36   NS  NS   80                          Pro--Asp--Ala--Arg--His--Ser--                                                                 241       103  103 92   100 ˜120                                                                        ˜130                   Thr--Thr--Gln--Pro--Arg                                                       Thr--Ile--Ser--Lys--Ala--Lys--                                                                 124       100  87  84   106 108 133                          Gly--Gln--Pro--Arg                                                            __________________________________________________________________________     NS indicates inhibition not significantly different from 0               

                                      TABLE 2                                     __________________________________________________________________________                          COUNTS/MINUTE                                                      CONCENTRATION                                                                            NO    PEPTIDE                                           TREATMENT  μg/ml   PEPTIDE                                                                             (100 μg/ml)                                                                       PEPTIDE INHIBITION                         __________________________________________________________________________    Culture medium only                                                                      --          228   343   -50                                        Concanavalin A                                                                           6.25        724   338   53                                         Concanavalin A                                                                           100.       5624  2989   47                                         (Succinylated)                                                                Phytohemagglutinin                                                                       6.25       3664  1897   48                                         Polkweed mitogen                                                                         0.05       2427  1413   42                                         Lens culinaris                                                                           25.        5460  2391   56                                         agglutinin                                                                    Vicia faba agglutinin                                                                    12.50      2200  1669   24                                         __________________________________________________________________________     Legend:                                                                       Effect of Thr--Ile--Ser--Lys--Ala--Lys--Gly--Gln--Pro--Arg on spontaneous     and lectininduced mitogenesis of human peripheral mononuclear cells.          Numbers shown are counts per minutes from .sup.125 I--Iododeoxyuridine        incorporation on day three of culture.                                   

                                      TABLE 3                                     __________________________________________________________________________    ROSETTE INHIBITION EXPRESSED AS PERCENT INHIBITION BY MONOMERIC IgG or        IgE                                                                                                     MONOCYTE/                                                            LYMPHOCYTE                                                                             MACROPHAGE                                                                             NEUTROPHIL                                                                              BASOPHIL                                          Fc Receptor                                                                            Fc Receptor                                                                            (PMN) Fc Receptor                                                                       Fc Receptor                      PEPTIDE          IgG  IgE IgG  IgE IgG       IgG IgE                          __________________________________________________________________________    Arg--Ser--Thr--Thr--Lys--Thr--                                                                 100  87  75   87  NS        90  110                          Ser--Gly--Pro--Arg                                                            Tyr--Ser--Lys--Leu--Thr--Val--                                                                 29   NS  NS   33  NS        NS  89                           Asp--Lys--Ser--Arg                                                            Asp--Lys--Ser--Arg                                                                             86   23  49   38  27        64  89                           Asp--Lys--Ser--Arg--Ala--Gln--                                                                 NS   NS  NS   NS  NS        NS  74                           Gln--Gly--Asn                                                                 Asp--Lys--Ser--Lys                                                                             16   NS  NS   NS  NS        NS  94                           __________________________________________________________________________     NS indicates inhibition not significantly different from 0               

                                      TABLE 4                                     __________________________________________________________________________    ROSETTE INHIBITION EXPRESSED AS PERCENT INHIBITION BY MONOMERIC IgG or        IgE                                                                                                              MONOCYTE/                                                    BASOPHIL                                                                             NEUTROPHIL                                                                              MACROPHAGE                                                                             LYMPHOCYTE                                          Fc Receptor                                                                          (PMN) Fc Receptor                                                                       Fc Receptor                                                                            Fc Receptor                       PEPTIDE           IgE IgG                                                                              IgG       IgG  IgE IgG  IgE                          __________________________________________________________________________    Thr--Arg--Ala--Glu--Ala--Glu--                                                                  108 NS NS        NS   21  NS   NS                           Glu--Lys--Asp                                                                 Thr--Arg--Ala--Glu                                                                              41  44 NS        24   20  49   NS                           Glu--Gln--Lys--Asp                                                                              106 18 NS        NS   NS  NS   NS                           Ser--Val--Met--His--Glu--Ala--                                                                  74  NS NS        NS   28  NS   NS                           Leu--His--Asn--His--Tyr--                                                     Thr--Gln--Lys                                                                 Asp--Ser--Asn--Pro--Arg                                                                         37  NS NS        NS   48  NS   NS                           __________________________________________________________________________     NS indicates inhibition not significantly different from 0               

Table 2 demonstrates representative immunostimulatory andimmunomodulatory activity of one of the subject peptides, viz.Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg when incubated with normal humanmononuclear cells (lymphocytes and monocytes) separated byficoll-metronidizole gradient. The cells were exposed to the peptide(final peptide concentration =100 micrograms/ml) followed by addition ofvarious plant-derived lectins (mitogens) which have well characterizedimmunomodulatory activity. Mitogenic lectins are thought to mimic someof the cellular events that accompany antigenic stimulation ofleukocytes and are useful indicators of cellular immune reactivity, invitro. Following lectin addition, the cells were cultured for severaldays and, at specified intervals, "pulsed" with radioactiveIododeoxyuridine which becomes incorporated into newly synthesized DNAof dividing cells.

After the cells were harvested and washed, the amount remainingcell-associated radioactivity may be used to estimate the degree towhich the cells were stimulated to proliferate. Table 2 shows that thepeptide strongly inhibited the mitogenic effects of the six mitogensindicating that the peptide can substantially modulate the proliferativeability of immunologically important mononuclear cells and thus has useas an immunomodulator. The peptide alone also stimulated mitogenesis(proliferation) of mononuclear cells by 50% on day 3, 331% on day 5 and283% on day 7. This indicates that the peptide can act as animmunostimulant by enhancing proliferation of mononuclear cells.

In the practice of the method of the present invention, an effectiveamount of a polypeptide or derivative thereof, or a pharmaceuticalcomposition containing same, as defined above, is administered via anyof the usual and acceptable methods known in the art, either singly orin combination with another compound or compounds of the presentinvention or other pharmaceutical agents such as antihistamines,corticosteroids, and the like. These compounds or compositions can thusbe administered orally, sublingually, topically (e.g. on the skin or inthe eyes), parenterally (e.g., intramuscularly, intravenously,subcutaneously or intradermally), or by inhalation, and in the form ofeither solid, liquid or gaseous dosage including tablets, suspensions,and aerosols, as discussed in more detail hereinafter. Theadministration can be conducted in single unit dosage form withcontinuous therapy or in single dose therapy ad libitum.

In one preferred embodiment, the method of the present invention ispracticed when the relief of symptoms is specifically required orperhaps imminent; in another preferred embodiment, the method hereof iseffectively practiced as continuous or prophylactic treatment.

In view of the foregoing as well as in consideration of the degree ofseverity of the condition being treated, age of subject, and so forth,all of which factors being determinable by routine experimentation byone skilled in the art, the effective dosage in accordance herewith canvary over a wide range. Since individual subjects vary in their Fcreceptor content, an effective systemic dosage in accordance herewithcan best be described as between 2×10³ and 2×10⁶ times the Fc receptorcontent, on a molar scale. For an average subject this would be betweenabout 0.5 and 500 mg/kg/day, depending upon the potency of the compound.Of course, for localized treatment, e.g., of the respiratory system,proportionately less material will be required.

Useful pharmaceutical carriers for the preparation of the compositionshereof, can be solids, liquids or gases; thus, the compositions can takethe form of tablets, pills, capsules, powders, enterically coated orother protected formulations (such as by binding on ion exchange resinsor other carriers, or packaging in lipid-protein vesicles or addingadditional terminal amino acids or replacing a terminal amino acid inthe L-form with one in the D-form), sustained release formulations,solutions (e.g., opthalmic drops), suspensions, elixirs, aerosols, andthe like. The carrier can be selected from the various oils includingthose of petroleum, animal, vegetable or synthetic origin, for example,peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water,saline, aqueous dextrose, and glycols are preferred liquid carriers,particularly (when isotonic) for injectable solutions. Suitablepharmaceutical excipients include starch, cellulose, talc, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk, glycerol, propylene glycol, water, ethanol,and the like. The compositions may be subjected to conventionalpharmaceutical expedients such as sterilization and may containconventional pharmaceutical additives such as preservatives, stabilizingagents, wetting or emulsifying agents, salts for adjusting osmoticpressure, buffers, and the like. Suitable pharmaceutical carriers andtheir formulation are described in "Remington's Pharmaceutical Sciences"by E. W. Martin. Such compositions will, in any event, contain aneffective amount of the active compound together with a suitable amountof carrier so as to prepare the proper dosage form for properadministration to the host.

To be effective for the prevention or treatment of the allergic reactionit is important that the therapeutic agents be relatively non-toxic,nonantigenic and non-irritating at the levels in actual use.

SYNTHESIS OF PEPTIDES

Peptides of this invention were synthesized by the solid phase peptidesynthesis (or Merrifield) method. This established and widely usedmethod, is described, including the experimental procedures, in thefollowing references:

Merrifield, J. Am. Chem. Soc., 85, 2149-2154 (1963).

Meinehofer in "Hormonal Proteins and Peptides," ed. C. H. Li, Vol. 2(Academic Press, 1973), pp. 48-267.

Barany and Merrifield in "The Peptides," eds. E. Gross and F.Meinenhofer, Vol. 2 (Adademic Press, 1980), pp. 3-285.

A preferred method for synthesizing the peptides of the presentinvention is the so-called "Merrifield" synthesis technique which iswell known to those skilled in the art and is set forth in detail in thearticle entitled "Synthesis of a Tetrapeptide" by R. B. Merrifield,Journal of the American Chemical Society (Vol. 85, pp. 2149-2154 (1963))as well as Meinehofer, cited above.

In this preferred method a peptide of any desired length and of anydesired sequence is produced through the stepwise addition of aminoacids to a growing peptide chain which is bound by a covalent bond to asolid resin particle.

In the preferred application of this method the C-terminal end of thegrowing peptide chain is covalently bound to a resin particle and aminoacids having protected amino groups are added in the stepwise mannerindicated above. A preferred amino protecting group is the t-BOC group,which is stable to the condensation conditions and yet is readilyremovable without destruction of the peptide bonds or racemization ofchiral centers in the peptide chain. At the end of the procedure thefinal peptide is cleaved from the resin, and any remaining protectinggroups are removed, by treatment under acidic conditions such as, forexample, with a mixture of hydrobromic acid and trifluoroacetic acid orwith hydrofluoric acid, or the cleavage from the resin may be effectedunder basic conditions, for example, with triethylamine, the protectinggroups then being removed under acid conditions.

The cleaved peptides are isolated and purified by means well known inthe art such as, for example, lyophilization followed by eitherexclusion or partition chromatography on polysaccharide gel media suchas Sephadex G-25, or countercurrent distribution. The composition of thefinal peptide may be confirmed by amino acid analysis after degradationof the peptide by standard means.

Salts of carboxyl groups of the peptide may be prepared in the usualmanner by contacting the peptide with one or more equivalents of adesired base such as, for example, a metallic hydroxide base, e.g.,sodium hydroxide; a metal carbonate or bicarbonate base such as forexample sodium carbonate or sodium bicarbonate; or an amine base such asfor example triethylamine, triethanolamine, and the like.

Acid addition salts of the polypeptides may be prepared by contactingthe polypeptide with one or more equivalents of the desired inorganic ororganic acid, such as, for example, hydrochloric acid.

Esters of carboxyl groups of the polypeptides may be prepared by any ofthe usual means known in the art for converting a carboxylic acid orprecursor to an ester. One preferred method for preparing esters of thepresent polypeptides, when using the Merrifield synthesis techniquedescribed above, is to cleave the completed polypeptide from the resinin the presence of the desired alcohol either under basic or acidicconditions, depending upon the resin. Thus the C-terminal end of thepeptide when freed from the resin is directly esterified withoutisolation of the free acid.

Amides of the polypeptides of the present invention may also be preparedby techniques well known in the art for converting a carboxylic acidgroup or precursor, to an amide. A preferred method for amide formationat the C-terminal carboxyl group is to cleave the polypeptide from asolid support with an appropriate amine, or to cleave in the presence ofan alcohol, yielding an ester, followed by aminolysis with the desiredamine.

N-acyl derivatives of an amino group of the present polypeptides may beprepared by utilizing an N-acyl protected amino acid for the finalcondensation, or by acylating a protected or unprotected peptide. O-acylderivatives may be prepared, for example, by acylation of a free hydroxypeptide or peptide resin Either acylation may be carried out usingstandard acylating reagents such as acyl halides, anhydrides, acylimidazoles, and the like. Both N- and O-acylation may be carried outtogether, if desired.

The coupling, deprotection/cleavage reactions and preparation ofderivatives of the subJect polypeptides are suitably carried out attemperatures between about -10° and +50° C., most preferably about20°-25° C. The exact temperature for any particular reaction will ofcourse be dependent upon the substrates, reagents, solvents and soforth, all being well within the skill of the practitioner. Illustrativereaction conditions for these processes may be gleaned from theexamples.

The following examples are given to enable those skilled in the art tomore fully understand and practice the present invention. They shouldnot be construed as a limitation upon the scope of the invention, butmerely as being illustrative and representative thereof.

EXAMPLE 1 Preparation of the 11-peptide (or undecapeptide)Thr-Val-Leu-His-Gln-Asn-Trp-Leu-Asp-Gly-Lys

5.3 gα-t-BOC-ε-Cl-Z-lysine resin (0.372 mole amino acid/g resin) wassubjected to a deprotection-neutralization cycle using the followingschedule:

(a) Three washes with methylene chloride

(b) Deprotection by treatment with 30% TFA (trifluoroacetic acid) inmethylene chloride for 20 minutes

(c) Three washes with methylene chloride

(d) Two washes with ethanol

(e) Three washes with methylene chloride

(f) Neutralization by 10% triethylamine in methylene chloride for 10minutes

(g) Three washes with methylene chloride

Threefold excess of the next amino acid in the sequence t-Boc-glycineand equivalent amount of dicyclohexyl carbodiimide in methylene chloridewere used to acylate the amino group of lysine. After two hours couplingtime a sample was removed to establish the completeness of the reactionby qualitative ninhydrin test. If incomplete, the coupling was repeatedas described above. Upon completion of the coupling reaction, thepeptide resin was washed three times with ethanol. The peptide resin wastaken through deprotection-neutralization cycles and the t-BOCderivative of the next amino acid (in the appropriate side chainprotected form) in sequence was coupled following the schedule describedfor the first amino acid.

Coupling of t-BOC-asparagine and t-BOC-glutamine was carried out in thepresence of equivalent amount of 1-hydroxybenztriazole. The attachmentof the last residue completed the synthesis. The peptide resin (7.3 g)was dried and treated with several drops of DMS (diamethylsulfide), 9 mlanisole and 70 ml liquid HF in an HF (hydrogen fluoride) apparatus. Thecleavage reaction proceeded for 60 minutes at 0° C. after which the HFwas removed under reduced pressure. The remaining material was washedwith ether, and was extracted with 60 ml of 50% acetic acid and 240 mlof water. Lyophilization gave 2.2 g crude, deprotected undecapeptide.

The first step of purification was by CCD (counter current distribution)using the solvent system n:butanol:acetic acid:water:ethanol in4:1:5:0.02 ratio. After 200 transfers tubes 39-59 were pooled to yield1.2 g material. It showed some tailing by TLC (thin layerchromatography) in the solvent system acetic acid:ethylacetate:water:n:butanol 1:1:1:1. This product was further purified on SG50 column by partition chromatography and eluted with the upper phase ofthe solvent system n-butanol:acetic acid:water 4:1:5. One hundredfifty-six (156) mg pure peptide was secured which proved to be pure byHPLC, TLC, amino acid analysis and paper electrophoresis.

Each of the peptides of the present invention may be prepared by ananalogous procedure by the stepwise addition of the desired amino acidto the growing peptide chain which is bound by a covalent bond to thesolid resin.

EXAMPLE 2 Preparation of Pro-Asp-Ala-Arg-His-Ser

5 g of α-t-BOC-Ser (OBzl) resin (0.46 mmole amino acid/g resin) wassubjected to deprotection, neutralization and coupling cycles followingthe schedule described for Example 1. Four molar excess of dicyclohexylcarbodiimide and the protected amino acids histidine, arginine, alanine,aspartic acid and proline were used in the subsequent coupling steps.

The completeness of the coupling reactions were established by thesemi-quantitative ninhydrin test on a small sample. After completion ofthe last coupling reaction, the dry peptide resin (6.8 g) was placedinto the reaction vessel of the HF cleavage apparatus and was treatedwith 60 ml of liquid HF and 6 ml of anisole at 0° C. for 60 minutes.After extraction and lyophilization 1.5 g crude peptide was isolated.The entire batch was applied to purification by CCD (counter currentdistribution) using the solvent system n-butanol:acetic acid:water in4:1:5 ratio. After 200 transfers 1.35 g material was collected fromtubes 6-24. This product was found to be pure by thin layerchromatogtaphy, high pressure liquid chromatography, paperelectrophoresis and amino acid analysis.

EXAMPLE 3 Preparation of the tetradecapeptideSer-Val-Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys

3.65 g α-t-BOC-ε-Cl-Z-lysine resin (0.414 mmole/g resin) was put throughdeprotection and neutralization cycles as described for EXAMPLE 1. Fourequivalents of t-BOC-glutamine, DCC (dicyclohexylcarbodiimide) and1-Hydroxybenztriazole were used in the next coupling cycle. Theappropriately protected amino acid residues corresponding to the primarysequence were coupled in subsequent steps to give 7.4 g of the protectedtetradecapeptide resin. HF cleavage was effected by addition of 5.0 mlanisole, a few drops of dimethylsulfide and 40 ml of liquid HF andstirring the mixture at 0° C. for 60 minutes. The HF was removed and theresidual material was washed with diethyl ether and extracted with 60 mlof 50% aqueous acetic acid followed by 240 ml water. The solution waslyophilized to give 1.8 g crude material, which was submitted topurification by counter current distribution using the solvent systemn-butanol:acetic acid:water in ratio 4:1:5 in 270 transfers. The mainfraction (tubes 26-37) were further purified by carboxymethyl celluloseion exchange chromatography with a linear gradient (0.01-0.3M) ofammonium acetate buffer of pH 4.5. The material corresponding to themain peak was purified by partition chromatography on a Sephadex G25 gelcolumn which provided the final product. Purity of the tetradecapeptidewas determined by HPLC, TLC, paper electrophoresis and amino acidanalysis.

EXAMPLE 4 Purification of Ac-Gln-Pro-Glu-Asn

After HF cleavage, the crude material (400 mg) was applied topurification by CCD using the solvent system n-butanol:acetic acid:waterin a 4:1:5 ratio. After 250 transfers, fractions 33-37 were pooled togive 53 mg of the pure product. This was found to be pure by thin layerchromatography, (solvent system nBuOH:Pyr:ACOH:H₂ O, 1:1:1:1) highpressure liquid chromatography, paper electrophoresis and amino acidanalysis.

EXAMPLE 5 Purification of Thr-Arg-Ala-Glu

The HF cleaved crude Thr-Arg-Ala-Glu (300 mg) was purified by a G-25partition column using n-BuOH:HOAc:H₂ O 4:1:5 upper layer as the eluentto afford partially purified Thr-Arg-Ala-Glu (163 mg). This peptide wasfurther purified by reversed phase liquid chromatography (C-18, 40micron) using 0.1% HOAc as the eluting solvent to afford pureThr-Arg-Ala-Glu (54 mg).

EXAMPLE 6 Purification of Glu-Lys-Gln-Arg

Crude peptide was purified by counter-current distribution usingn-BuOH:HOAc:H₂ O (4:1:5) system to afford Glu-Lys-Gln-Arg. This peptidewas purified by reversed phase liquid chromatography (C-18, 40 micron)using 0.1% HOAc to give the pure Glu-Lys-Gln-Arg.

EXAMPLE 7 Purification of Arg-Ser-Thr-Thr-Lys-Thr-Ser-Gly-Pro-Arg

The dry peptide resin (4.1 g) was placed into the reaction vessel of theHF cleavage apparatus and was treated with 40 ml of liquid HF and 5.0 mlof anisole at 0° C. for 60 minutes. After extraction and lyophilization750 mg crude peptide was isolated.

The entire batch was purified by CCD (counter space currentdistribution) using the solvent system n-butanol:acetic acid:water in4:1:5 ratio. After 220 transfers, fractions 81-85 were pooled to give200 mg of the pure product. This product was purified by thin layerchromatography (solvent system n-butanol:Pyr:acetic acid:water,15:10:3:12), and confirmed by high pressure liquid chromatography, paperelectrophoresis, and amino acid analysis.

EXAMPLE 8 Purification of Asp-Lys-Ser-Arg

The dry peptide resin (7.3 g) was placed into the reaction vessel of theHF cleavage apparatus and was treated with 60 ml of liquid HF and 8 mlanisole at 0° C. for 60 minutes. After extraction and lyophilizationapproximately 1 g crude peptide was isolated.

The entire batch was purified by CCD (counter current distribution)using the solvent system n-butanol:acetic acid:water in 4:1:5 ratio.After 220 transfers, fractions 65 to 110 were pooled to giveapproximately 1 g of product. The product was purified by liquidchromatography using a cation exchange column and partition columns. Thepurified product was confirmed by amino acid analysis.

As used in the present specification and claims an active site peptideis a peptide with amino acid sequence derived from an immunoglobulinactive site. An immunoglobulin active site is a portion of animmunoglobulin molecule which physically binds to an immunoglobulin Fcreceptor, thereby triggering Fc receptor functions. An active sitepeptide, by virtue of its resemblance to a portion of an intactimmunoglobulin molecule, is able to bind to an immunoglobulin Fcreceptor and thereby mimic the intact immunoglobulin. Such binding mayeither stimulate or inhibit the specific immune function normallymediated by the receptor.

It is understood that various other modifications will be apparent toand can readily be made by those skilled in the art without departingfrom the scope and spirit of this invention. Accordingly, it is notintended that the scope of the claims appended hereto be limited to thedescription as set forth above, but rather that the claims be construedas encompassing all the features of patentable novelty which reside inthe present invention, including all features which would be treated asequivalents thereof by those skilled in the art to which this inventionpertains.

What is claimed is:
 1. A method of modulating an immune response in amammal by blocking immune complex binding to immunoglobulin Fcreceptors, said method comprising administering an effective amount of apeptide having the amino acidsequenceA-Pro-Asp-Ala-Arg-His-Ser-Thr-Thr-Gln-Pro-Arg-B,wherein A is asubstituent selected from the group consisting of H-, Gln-, Leu-Gln-,Val-Leu-Gln-, and Glu-Val-Leu-Gln-;and wherein B is a substituentselected from the group consisting of -OH, -Lys, -Lys-Thr, and-Lys-Thr-Lys;and pharmaceutically acceptable salts of said peptide.
 2. Amethod of modulating an immune response in a mammal by blocking immunecomplex binding to immunoglobulin Fc receptors, said method comprisingadministering an effective amount of a peptide having the amine acidsequenceC-Thr-Thr-Gln-Pro-Arg-D,wherein C is a substituent selected fromthe group consisting of H-, Ser-, His-Ser-, Arg-His-Ser-,Ala-Arg-His-Ser-, and Asp-Ala-Arg-His-Ser-;and wherein D is asubstituent selected from the group consisting of -OH, -Lys, -Lys-Thr,and -Lys-Thr-Lys;and pharmaceutically acceptable salts of said peptide.3. A method of modulating an immune response in a mammal by blockingimmune complex binding to immunoglobulin Fc receptors, said methodcomprising administering an effective amount of a peptide having theamino acid sequenceThr-Gln-Pro-Arg-E,wherein E is a substituent selectedfrom the group consisting of -OH, -Lys, -Lys-Thr, and -Lys-Thr-Lys;andpharmaceutically acceptable salts of said peptide.
 4. A method ofmodulating an immune response in a mammal by blocking immune complexbinding to immunoglobulin Fc receptors, said method comprisingadministering an effective amount of a peptide selected from the groupconsisting ofArg-His-Ser-Thr-Thr-F Ala-Arg-His-Ser-G, Asp-Ala-Arg-His-J,and K-Pro-Asp-Ala-Arg,wherein F is a substituent selected from the groupconsisting of -OH, -Gln, -Gln-Pro, -Gln-Pro-Arg, -Gln-Pro-Arg-Lys,-Gln-Pro-Arg-Lys-Thr, and -Gln-Pro-Arg-Lys-Thr-Lys;wherein G is asubstituent selected from the group consisting of -OH -Thr, and-Thr-Thr-F;wherein J is a substituent selected from the group consistingof -OH, -Ser, and -Ser-G;and wherein K is a substituent selected fromthe group consisting of H-, Leu-, Gln-Leu, Val-Gln-Leu, andGlu-Val-Gln-Leu;and pharmaceutically acceptable salts of said peptide.5. The method of modulating an immune response by blocking immunecomplex binding as set forth in claim 1, comprising administering aneffective amount of said peptide to reduce immune complex mediatedinflammation or tissue destruction.
 6. The method of modulating animmune response by blocking immune complex binding as set forth in claim1, comprising administering an effective amount of said peptide toreduce the human allergic response.
 7. The method of modulating animmune response by blocking immune complex binding as set forth in claim2, comprising administering an effective amount of said peptide toreduce immune complex mediated inflammation or tissue destruction. 8.The method of modulating an immune response by blocking immune complexbinding as set forth in claim 2, comprising administering an effectiveamount of said peptide to reduce the human allergic response.
 9. Themethod of modulating an immune response by blocking immune complexbinding as set forth in claim 3, comprising administering an effectiveamount of said peptide to reduce immune complex mediated inflammation ortissue destruction.
 10. The method of modulating an immune response byblocking immune complex binding as set forth in claim 3, comprisingadministering an effective amount of said peptide to reduce the humanallergic response.
 11. The method of modulating an immune response byblocking immune complex binding as set forth in claim 4, comprisingadministering an effective amount of said peptide to reduce immunecomplex mediated inflammation or tissue destruction.
 12. The method ofmodulating an immune response by blocking immune complex binding as setforth in claim 4, comprising administering an effective amount of saidpeptide to reduce the human allergic response.